Methods for starch hydrolysis

ABSTRACT

The presently disclosed subject matter provides a process for starch liquefaction using at least two classes of α-amylase enzymes, wherein the starch hydrolysis pattern from at least two of these classes is different. At least one class of enzyme is provided to the liquefaction process in the form of transgenic plant material expressing at least one class of α-amylase enzyme or is provided in the form of a purified or partially-purified α-amylase enzyme preparation. The second or subsequent class(es) of α-amylase enzymes may be provided in the form of additional transgenic plant material expressing the second or subsequent class(es), or may be provided in the form of a second or subsequent purified or partially-purified α-amylase enzyme preparation.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. patent application Ser. No.12/395,180 filed Feb. 27, 2009 (allowed), which claims the benefit ofU.S. Provisional Patent Application No. 61/032,773 filed Feb. 29, 2008,the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to processing starch from plant sources,particularly for the production of ethanol.

BACKGROUND OF THE INVENTION

Starch is a complex carbohydrate often found in the human diet. Plantsare often used as a source for starch, which can be used to produceethanol and other products. Plant starches are generally in a granularform, which is insoluble in water. Conventional plant starch processingmethods often involve a starch gelatinization process, wherein aqueousstarch slurry is heated so that the granular starch in the slurry swellsand bursts, dispersing starch molecules into the solution. During thegelatinization process, there is a dramatic increase in viscosity. Toenable handling during the remaining process steps, the starch must bethinned or “liquefied”. This reduction in viscosity can be accomplishedby enzymatic degradation in a process referred to as liquefaction.During liquefaction, the long-chained starch molecules are degraded intosmaller branched and linear chains of glucose units (dextrins) by anenzyme, such as α-amylase (i.e., α-amylase).

Amylase is an enzyme that catalyzes the hydrolysis of starches intosugars. Amylases hydrolyze internal α-1,4-glucosidic linkages in starch,largely at random, to produce smaller molecular weight maltodextrins.Amylases are of considerable commercial value, as they are used in theinitial stages (liquefaction) of starch processing; in wet corn milling;in alcohol production; as cleaning agents in detergent matrices; in thetextile industry for starch desizing; in baking applications; in thebeverage industry; in oilfields in drilling processes; in inking ofrecycled paper; and in animal feed.

SUMMARY OF THE INVENTION

The presently disclosed subject matter provides a process for starchliquefaction and the production of ethanol using at least two classes ofα-amylase enzymes, wherein the starch hydrolysis pattern from at leasttwo of these classes is different. In some embodiments, at least oneclass of enzyme is provided to the liquefaction process in the form oftransgenic plant material expressing at least one class of α-amylaseenzymes. The second or subsequent classes of α-amylase enzymes may beprovided in the form of additional transgenic plant material expressingthe second or subsequent class(es), or may be provided in the form of apurified or partially-purified α-amylase enzyme preparation.Alternatively, two or more classes of α-amylase enzymes may be providedas purified or partially-purified α-amylase enzyme preparations.

Also provided is a process for the production of ethanol comprising,liquefying an aqueous slurry of starch-containing plant material in thepresence of at least two classes of α-amylase enzymes, wherein thestarch hydrolysis pattern from at least two of these classes isdifferent, and using the liquefact in a fermentation process to generateethanol.

One embodiment of the invention is a process for starch liquefaction,the process comprising liquefying an aqueous slurry of starch-containingplant material in the presence of at least a first and a second class ofα-amylase enzymes, wherein the first class of α-amylase enzymes exhibitsa starch hydrolysis pattern that is different from the starch hydrolysispattern of at least the second class of α-amylase enzymes. The followingembodiment may be carried out wherein the starch-containing plantmaterial comprises a transgenic plant part comprising a polynucleotideencoding either the first or second class of α-amylase enzymes or thetransgenic plant may be engineered to express both enzymes. The processmay also comprise a purified or partially-purified first and secondclass α-amylase enzyme, wherein the process comprises one or moreliquefaction steps that are performed under conditions sufficient forthe first and second class α-amylase enzymes to hydrolyze saidstarch-containing plant material. In another embodiment, it may bebeneficial to add the first and second class enzymes into the slurry ina liquefaction step that is performed under conditions sufficient forthe first and second classed of α-amylase enzymes to hydrolyze saidstarch-containing plant material. In some embodiments thestarch-containing plant material is derived from a plant selected fromthe group consisting of rice, barley, potato, sweet potato, canola,sunflower, rye, oats, wheat, corn, soybean, sugar beet, tobacco,Miscanthus grass, Switch grass, safflower, trees, cotton, cassava,tomato, sorghum, alfalfa and sugarcane.

Another embodiment presented in the current invention is a process forproducing ethanol comprising liquefying an aqueous slurry ofstarch-containing plant material in the presence of at least a first anda second class of α-amylase enzymes, wherein the first class ofα-amylase enzymes exhibits a starch hydrolysis pattern that is differentfrom the starch hydrolysis pattern of at least the second class ofα-amylase enzymes to obtain a liquefact; and, fermenting the liquefactby yeast to obtain ethanol. The process may further comprise asaccharification step in the presence of a glucoamylase, wherein thesaccharification step can be performed simultaneously with liquefactionand fermentation.

Another embodiment is a process for producing ethanol comprising rawstarch fermentation of starch-containing plant material in the presenceof at least a first and a second class of α-amylase enzymes, wherein thefirst class of α-amylase enzymes exhibits a starch hydrolysis patternthat is different from the starch hydrolysis pattern of at least thesecond class of α-amylase enzymes.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention now will be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the inventions are shown. Indeed, theseinventions may be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will satisfy applicablelegal requirements. Like numbers refer to like elements throughout.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element. Throughout thespecification the word “comprising,” or variations such as “comprises”or “comprising,” will be understood to imply the inclusion of a statedelement, integer or step, or group of elements, integers or steps, butnot the exclusion of any other element, integer or step, or group ofelements, integers or steps.

Overview

The first step in the processing of grain to ethanol in the dry grindprocess involves the hydrolysis of starch mediated by α-amylases.Typical corn-to-ethanol conversion processes utilize an α-amylase enzymederived from a bacterium of the genus Bacillus to liquefystarch-containing plant material (e.g., grain) in the presence of water.The liquefaction reaction involves heating a combination of ground grainand water beyond the grains' gelatinization point under slightly acidicconditions in the presence of an enzyme that will hydrolyze the linkagebetween the glucose units rendering a complex mixture of dextrins,sugars and other retrograde products.

Most α-amylase enzymes from the Bacillus genus have a pattern ofhydrolysis yielding a characteristic composition of these breakdownproducts (i.e., “hydrolysates”). Another class of α-amylase enzymes hasa novel pattern of starch hydrolysis and produces a very differentcombination of hydrolysates. The performance in terms of ethanolproduction and residual sugars/starch of the liquefied substrateproduced with either class of α-amylases is very similar. Both classesof alpha-amylase enzymes are able to hydrolyse starch in a manner thatsupports ethanol production. However, while not bound by any particulartheory or mechanism, it is believed that using the two different classesof α-amylase enzymes together in a liquefaction reaction will produce asubstrate that will give higher concentrations of ethanol and lessresidual sugars/starch in a fermentation process than the use of eitherclass alone.

Thus, described herein are methods for the liquefaction ofstarch-containing plant material using at least two different classes ofα-amylase enzymes, wherein the process comprises liquefying an aqueousslurry of starch-containing plant material in the presence of at least afirst and a second class of α-amylase enzymes, wherein the first classof α-amylase enzymes exhibits a starch hydrolysis pattern that isdifferent from the starch hydrolysis pattern of at least the secondclass of α-amylase enzymes. By “starch hydrolysis pattern” is intendedthe collection of hydrolysates resulting from the enzymatic conversionof starch to sugar. While the activity of α-amylase is primarilyhydrolytic, several amylase enzymes are known to have transglycosylationactivity, resulting in the production of higher molecular weightoligosaccharides (see, for example, Thompson et al. (1997) Nucleic AcidsRes. 25:4876-4882 and Rivera et al. (2003) Protein Engineering16(7):505-514). Thus, for the purposes of the present invention, thestarch hydrolysis pattern includes sugars resulting from hydrolysis aswell as from transglycosylation.

In various embodiments, the first class of α-amylase, the second classof alpha amylase, or both, is provided in the form of transgenic plantmaterial. In another embodiment, either the first or the second class ofα-amylase enzymes, or both, is provided as a purified orpartially-purified preparation of the α-amylase enzyme. In yet anotherembodiment, one class of α-amylase is provided as transgenic plantmaterial expressing the α-amylase, and the other α-amylase is providedexogenously to the slurry as a purified or partially-purifiedpreparation of the enzyme. It is to be understood that the terms “first”and “second” are used for purposes of distinguishing two differentclasses of enzymes, but is not related to the order in which each isused in a starch-conversion process.

Alpha-Amylase

The present invention relates to methods of starch hydrolysis using atleast two different classes of alpha-amylase (“α-amylase”) enzymes. Asused herein, the term “amylase” encompasses enzymes (e.g., E.C. class3.2.1.1) having α-amylase activity, for example, α-amylases capable ofhydrolyzing internal α-1,4-glucan links in polysaccharides, includingamylase enzymes capable of hydrolyzing starch to sugars at alkaline pHsor at acidic pHs. These enzymes have also been described as thoseeffecting the exohydrolysis or endohydrolysis of 1,4-α-D-glucosidiclinkages in polysaccharides containing 1,4-α-linked D-glucose units.Another term used to describe these enzymes is “glycogenase.”

The α-amylase enzymes useful herein are characterized and classifiedaccording to the number and/or type of hydrolysis products resultingfrom liquefaction of starch-containing plant material in the presence ofthe α-amylase enzyme. For the purposes of the present invention, thetype of hydrolysis product refers to the degree of polymerization (DP)of the product. For example, DP1 and DP2 refer to mono- anddisaccharides, respectively; DP3 to DP9 refer to oligosaccharidescontaining three to nine monosaccharide units; and designations of DP10or greater refer to polysaccharides containing 10 or more monosaccharideunits. Hydrolysis products are considered to be in the “lower sizerange” if the DP is equal to or less than 30. Hydrolysis products areconsidered to be “higher size range” if the DP is greater than 30.

The starch hydrolysis pattern for an α-amylase enzyme can be determinedusing any number of techniques. In one embodiment, the hydrolysispattern is characterized by analyzing the hydrolysis products using sizeexclusion chromatography as described in the Experimental Examplesherein. In another embodiment, the hydrolysis pattern can be determinedbased on the method described in Robyt & French (1967) Archives ofBiochemistry and Biophysics 100:451-467, optionally as modified inAtichokudomchai et al. (2006) Carbohydrate Polymers 64:582-588, or inMacGregor et al. (1992) Carbohydrate Res. 227:301-313; Nakakuki et al.Carbohydrate Res. 128 (1984) 291-310; Saito, N. Archives of Biochemistryand Biophysics 155, 290-298 (1973); Marchal et al. Biotechnology andBioengineering, Vol 63, No. 3, May 5, 1999; Ivanova, et al. AppliedBiochemistry and Biotechnology Vol 30, 1991. 193-202; and, Heitmann etal. Enzyme and Microbial Technology 20:259-267, 1997, each of which isherein incorporated by reference in its entirety.

The hydrolysis pattern is typically measured within a particulardextrose equivalent range. Dextrose equivalent (DE) is the industrystandard for measuring the concentration of total reducing sugars,calculated as D-glucose on a dry weight basis. Un-hydrolyzed granularstarch has a DE of virtually zero, whereas the DE of D-glucose isdefined as 100. In one embodiment, the hydrolysis pattern is measuredwhen the DE is between 10 and 20, or between 10 and 15.

In the methods of the present invention, liquefaction of thestarch-containing plant material is performed in the presence of atleast two classes of α-amylase enzymes. In one embodiment, at least oneclass of α-amylase enzymes exhibits a starch hydrolysis pattern that isunimodal in distribution, and the other class exhibits a starchhydrolysis pattern that is bimodal in distribution. In anotherembodiment, liquefaction is performed in the presence of at least twodifferent classes of α-amylase enzymes, wherein each class exhibits adifferent unimodal distribution. A “unimodal” distribution occurs whensubstantially all of the detectable hydrolysis products fall within aparticular size range (e.g., higher or lower size ranges). Where themethod employs at least two different unimodal classes of α-amylaseenzymes, substantially all of the hydrolysis products from one classwill fall into one size range, and substantially all of the hydrolysisproducts from the other class will fall into a different size range. By“substantially all” is intended at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or greater, of the detectable hydrolysis productsfall within a particular size range. A “bimodal” distribution occurswhen the hydrolysis products fall into at least 2 different size ranges.To be considered “bimodal,” greater than 10% of the total detectablehydrolysis product must be represented in at least two different sizeranges (i.e., at least 10% must be in one size range, and at least 10%must be in a different size range).

It is also contemplated that α-amylase enzymes will be identified thatexhibit “multimodal” distribution patterns, where at least 10% of thetotal detectable hydrolysis product is represented in more than 2 sizeranges, may exhibit a bimodal pattern with at least 10% of the totaldetectable hydrolysis products present in at least 2 size ranges thatare different from the “high” and “low” patterns defined supra. In theseembodiments, the size ranges may include, for example, any range fromDP1 to greater than DP4000 (e.g., DP less than 10, DP less than 20, DPless than 40, DP less than 50, DP less than 100, DP1 to DP10, DP1-DP5,DP5-DP15, DP10-DP20, DP15-DP25, DP20-DP40, DP25-DP45, DP40-DP60,DP45-DP65, DP60-DP100, DP70-DP110, DP100-DP200, DP200-DP500, DP500-1000,DP1000-DP2000, DP2000-DP3000, DP3000-DP4000, DP greater than 500, DPgreater than 600, DP greater than 700, DP greater than 1000, DP greaterthan 2000, DP greater than 3000, and DP greater than 4000). Suchmultimodal and alternative bimodal classes of α-amylase enzymes areuseful in the present invention. However, unless otherwise specified,the term “bimodal” refers to a starch hydrolysis pattern in which atleast 10% of the total detectable products are in the lower size rangedefined by DP less than or equal to 30, and at least 10% of thedetectable products are in the higher size range defined by DP greaterthan 30.

Suitable α-amylases include naturally occurring α-amylases as well asrecombinant or mutant amylases which are useful in liquefaction ofstarch. In one embodiment, the class of α-amylase enzymes exhibiting aunimodal starch hydrolysis pattern includes the α-amylase (“797GL3”)described in Richardson et al. (2002) J Biol Chem. 277(29):26501-7 whichis herein incorporated by reference in its entirety. In anotherembodiment, the unimodal α-amylase is the α-amylase (“D45”) described inAtichokudomchai et al. (2006) Carbohydrate Polymers 64:582-588, hereinincorporated by reference in its entirety. See also, US PatentPublication 2003/0125534 and U.S. Patent Publication 2004/0018607 (bothare herein incorporated by reference) which describe numerous otherα-amylase enzymes that share sequence identity to the 797GL3, BD12870and D45 enzymes. In another embodiment it is expected that α-amylasesderived from the microorganism order Thermococcales demonstrate aunimodal hydrolysis pattern.

Suitable classes of α-amylase enzymes exhibiting a bimodal distributionof hydrolysis products include, for example, the α-amylases derived fromBacillus sp. (e.g., Bacillus licheniformis and Bacillusamyloliquefaciens α-amylase enzymes).

The hydrolysis pattern can also be determined for any number of knownamylase enzymes using the methods described herein. Amylases areproduced by a wide variety of microorganisms including Bacillus andAspergillus, with most commercial amylases being produced from bacterialsources such as Bacillus licheniformis, Bacillus amyloliquefaciens,Bacillus subtilis, or Bacillus stearothermophilus. Amylase enzymesfalling into a desirable hydrolysis “class” can then be used in themethods of the invention.

Techniques for producing variant amylases are also known in the art.Such techniques could be utilized to alter the hydrolysis properties ofknown amylase enzymes to suit the needs of the present invention.

Additionally, polynucleotides encoding the characterized α-amylasesdescribed herein or otherwise known in the art may be used to isolatehomologous sequences from cultured organisms or environmental samples.In one embodiment, gene libraries generated from one or more α-amylaseexpressing microorganisms can be screened for amylase enzymes exhibitinga particular hydrolysis pattern. Methods for making and using organismsexpressing α-amylase enzymes (for example, to produce fermentablesubstrates for the production of ethanol) are also provided in U.S.Patent Publication No. 2003/0135885, which is herein incorporated byreference in its entirety.

Enzyme Extracts

In various embodiments of the present invention, either the first or thesecond class of α-amylase enzymes, or both, is provided as a crude,purified or partially-purified preparation of the α-amylase enzyme. Theexogenously-added α-amylase enzyme may be de novo synthesized, or may beisolated from an organism expressing the α-amylase enzyme prior toaddition of the enzyme to the starch-containing plant material, or maybe through the addition of a crude extract containing at least oneenzyme useful in starch conversion.

An exogenously-added enzyme may be a crude, purified orpartially-purified preparation of enzyme containing at least one classof α-amylase enzyme, but may also contain one or more additionalα-amylase enzymes of the same or different class. The preparation mayfurther comprise one or more additional enzymes useful in the starchconversion method, such as glucoamylase. A “partially-purified” enzymepreparation will contain one or more α-amylase enzymes, one or moreadditional enzymes useful in the starch conversion process, or maycontain other buffers or stabilizing agents (e.g., glycerol).Furthermore, the partially-purified enzyme preparation may also beculture supernatant or crude extract collected from a cell populationexpressing and/or secreting the enzyme. The preparation may also be alyophilized formulation of enzyme that is reconstituted upon addition tothe starch-containing plant material.

Alpha-amylase enzymes can be expressed in and isolated from any numberof eukaryotic and prokaryotic organisms. Appropriate expressioncassettes, vectors, transformation, and transfection techniques for aparticular organism of interest will be evident to one of skill in theart.

In one embodiment, bacterial cells, such as E. coli, Streptomyces,Bacillus subtilis; and various species within the genera Escherichia,Pseudomonas, Serratia, Streptomyces, Corynebacterium, Brevibacterium,Bacillus, Microbacterium, and Staphylococcus can be used as a host toexpress one or more classes of α-amylase enzymes encompassed herein.Methods for transformation of bacterial hosts are described in, forexample, U.S. Patent Publication No. 2003/0135885.

In another embodiment, fungal hosts, such as fungal host cells belongingto the genera Aspergillus, Rhizopus, Trichoderma, Neurospora, Mucor,Penicillium, etc., such as yeast belonging to the genera Kluyveromyces,Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, etc.may be used. Transformation of fungus may be accomplished according toGonni et al. Agric. Biol. Chem., 51:2549 (1987).

Another suitable host includes any number of eukaryotic cells, forexample, insect cells such as Drosophila S2 and Spodoptera Sf9; animalcells such as CHO, COS or Bowes melanoma, C127, 3T3, CHO, HeLa and BHKcell lines. Any host can be used insofar as it can express the gene ofinterest. The American Type Culture Collection (http://www.atcc.org/)maintains cell lines from a wide variety of sources and many of thesecultures can be used to generate a transgenic cell line capable ofexpressing an α-amylase enzyme. Transformation vectors appropriate foreukaryotic cells are available commercially such as pXT1, pSG5(Stratagene) pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Techniques fortransformation and selection of transgenic eukaryotic cells are wellknown in the art.

Additional methods for generating an enzyme extract are described in,for example, Conrad et al. (1995) Eur. J. Biochem. 230, 481-490; Chianget al. (1979) Starch 31 Nr. 3, S. 86-92; Schwardt, E. (1990) FoodBiotechnology, 4(1), 337-351; Morgan and Priest (1981) Journal ofApplied Bacteriology 50, 107-114; Laderman et al. (1993) Journal ofBiological Chemistry Vol. 268, No. 32, pp. 24394-24401, each of which isherein incorporated by reference in its entirety.

Transgenic Plants

In one embodiment of the present invention, the starch-containing plantmaterial comprises plant parts derived from at least one variety of atransgenic plant expressing a polynucleotide encoding an α-amylaseenzyme. As used herein the term “transgenic” refers to plants thatinclude an exogenous polynucleotide (e.g., gene) that is stablymaintained in the transformed plant and is stably inherited by progenyin successive generations. The term “transgenic plant” can refer eitherto the initially transformed plant or to the progeny of the initiallytransformed plant. Techniques for transforming plants, plant cells orplant tissues can include, but are not limited to, transformation withDNA employing A. tumefaciens or A. rhizogenes as the transforming agent,electroporation, DNA injection, microprojectile bombardment, andparticle acceleration. See, for example, EP 295959 and EP 138341. Asused herein, the terms “plant material” or “plant part” includes plantcells, plant protoplasts, plant cell tissue cultures from which plantscan be regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants such as embryos, pollen, ovules,seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks,stalks, roots, root tips, anthers, tubers, rhizomes and the like. Asused herein, the term “plant part” or “plant tissue” includes plantcells, plant protoplasts, plant cell tissue cultures from which plantscan be regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants such as embryos, pollen, ovules,seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks,stalks, roots, root tips, anthers, and the like.

In this embodiment, it is not necessary for the starch-containing plantmaterial to be 100% transgenic for the α-amylase enzyme. Rather, it isonly necessary for the plant material to contain an amount of amylasethat is sufficient for the downstream use. For example, for fermentationpurposes, a sufficient amount of amylase enzyme may be provided in thefermentation process by less than 100% amylase-expressing plantmaterial. For example, a sufficient amount of amylase enzyme may beprovided to the fermentation process when only about 0.1% of the plantmaterial expresses amylase, or only about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, or about 20%, of the plant material. However, itis contemplated that the percentage of plant material expressing theα-amylase could be as much as 100%, including, for example, about 25%,about 30%, about 35%, about 40%, about 50%, about 60%, about 65%, about70%, about 80%, about 90%, about 95%, or about 99% of the plantmaterial.

Where both classes of α-amylase enzymes are provided as transgenic plantmaterial, each class of α-amylase may be expressed in the same plantvariety, or may be expressed in different plant varieties. Where eachamylase is expressed in different plant varieties, the transgenic plantmaterial can be combined at a target ratio necessary to achieve a targetlevel of each of the α-amylase enzymes necessary for optimalliquefaction. “Optimal liquefaction” can refer to the total amount ofstarch-derived product, as well as the variety of differentstarch-derived products resulting from hydrolysis of thestarch-containing plant material. For example, the starch-derivedproducts can include oligo- and polysaccharides having various chainlengths or having various branching patterns.

The amount of the starch-containing transgenic plant material can beadjusted to effect optimal liquefaction (i.e., by adjusting the amountof starch-digesting enzyme provided in the slurry). The amount can varydepending upon the plant material, the desired mixture of products inthe starch liquefact, the desired speed of liquefaction, or upon apre-selected liquefaction temperature. Optimal liquefaction willtypically be determined by the downstream user (e.g., ethanol producer)and takes into account a variety of factors including, but not limitedto, the level or expression of the amylase in the plant or plant part,the type of plant utilized, and processes involved in converting thestarch-containing plant material to a useful product (e.g., food, feed,industrial alcohol, biofuel, fermentation product, etc.). For example,an ethanol production facility interested in utilizing a combination ofα-amylase enzymes having different starch hydrolysis patterns, thetarget ratio necessary to achieve optimal liquefaction will take intoaccount the chemical conversion and/or fermentation processes involvedin converting the plant material to ethanol, including the reactionconditions, the level or activity of any exogenous enzymes (α-amylase orotherwise) that may be included in the process, the types of oligo- andpolysaccharides desirable for feedstock, as well as any other materialsrequired for each step in the conversion. One of skill in the art willunderstand how to determine the amount of amylase or amylase-expressingplant material to use for a particular downstream use.

Thus, in one embodiment, the starch-containing plant material comprisesabout 0.1% to about 99.9%, including about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, about 20%, 1%, a 21%, about 22%, about 23%, about24%, about 25%, about 30%, about 35%, 0%, a 40%, about 45%, about 50%,about 55, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, about 97%, about 98%, and about 99%, plantmaterial derived from plants expressing a first class of α-amylase andabout 0.1 to about 99.1%, including about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, %, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 97%, about 98%, and about 99%,plant material derived from plants expressing a second class ofα-amylase, wherein the sum of the percentage of each plant varietyequals 100% of the total plant material. The starch-containing plantmaterial may further comprise about 0.1% to about 99.8%, including about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%,about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%,about 98%, and about 99%, plant material derived from one or moreadditional varieties, which may or may not express an α-amylase enzyme(e.g., “wild-type” plant material, or plant material expressing one ormore transgenes other than α-amylase).

The transgenic or non-transgenic starch-containing plant material can bederived from any plant, including but not limited to plants producingedible flowers such as cauliflower (Brassica oleracea), artichoke(Cynara scolvmus), and safflower (Carthamus, e.g. tinctorius); fruitssuch as apple (Malus, e.g. domesticus), banana (Musa, e.g. acuminata),berries (such as the currant, Ribes, e.g. rubrum), cherries (such as thesweet cherry, Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus),grape (Vitis, e.g. vinifera), lemon (Citrus limon), melon (Cucumismelo), nuts (such as the walnut, Juglans, e.g. regia; peanut, Arachishypoaeae), orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica),pear (Pyra, e.g. communis), pepper (Solanum, e.g. capsicum), plum(Prunus, e.g. domestica), strawberry (Fragaria, e.g. moschata), tomato(Lycopersicon, e.g. esculentum); leafs, such as alfalfa (Medicago, e.g.sativa), sugar cane (Saccharum), cabbages (such as Brassica oleracea),endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce(Lactuca, e.g. sativa), spinach (Spinacia e.g. oleraceae), tobacco(Nicotiana, e.g. tabacum); roots, such as arrowroot (Maranta, e.g.arundinacea), beet (Beta, e.g. vulgaris), carrot (Daucus, e.g. carota),cassava (Manihot, e.g. esculenta), turnip (Brassica, e.g. rapa), radish(Raphanus, e.g. sativus) yam (Dioscorea, e.g. esculenta), sweet potato(Ipomoea batatas); seeds, such as bean (Phaseolus, e.g. vulgaris), pea(Pisum, e.g. sativum), soybean (Glycine, e.g. max), wheat (Triticum,e.g. aestivum), barley (Hordeum, e.g. vulgare), corn (Zea, e.g. mays),rice (Oryza, e.g. sativa); grasses, such as Miscanthus grass(Miscanthus, e.g., giganteus) and switchgrass (Panicum, e.g. virgatum);trees such as poplar (Populus, e.g. tremula), pine (Pinus); shrubs, suchas cotton (e.g., Gossypium hirsutum); and tubers, such as kohlrabi(Brassica, e.g. oleraceae), potato (Solanum, e.g. tuberosum), and thelike.

The starch-containing plant material may also comprise one or morevarieties of plants having naturally-occurring genetic variabilityresulting in altered starch metabolism. Many such plants carry mutationsin genes encoding isoforms of starch synthesis or starch degradationenzymes. For example, plants have been identified which are heterozygousor homozygous for one or more of the waxy (wx), amylose extender (ae),dull (du), horny (h), shrunken (sh), brittle (bt), floury (fl), opaque(o), or sugary (su) mutant alleles. See, for example, U.S. Pat. Nos.4,428,972; 4,767,849; 4,774,328; 4,789,738; 4,789,557; 4,790,997;4,792,458; 4,798,735; and 4,801,470, herein incorporated by reference.These plants can be used in their native form, or can be modified toexhibit one or more transgenes (e.g., amylase, or other commercially oragronomically useful transgenes) of interest.

Methods

The methods of the present invention are directed to a process forstarch liquefaction, the process comprising liquefying an aqueous slurryof starch-containing plant material in the presence of at least a firstand a second class of α-amylase enzyme, wherein the first class ofα-amylase enzyme exhibits a starch hydrolysis pattern that is differentfrom the starch hydrolysis pattern of at least the second class ofα-amylase enzyme. The term “slurry” refers to a mixture of starch or astarch-containing material (e.g., milled corn) and an aqueous component,which can include, for example, water, deionized water, or a processwater (i.e., backset, steam, condensate), or any combination thereof. Asused herein the terms “liquefaction,” “liquefy,” “liquefact,” andvariations thereof refer to the process or product of converting starchto soluble dextrinized substrates (e.g., smaller polysaccharides).Liquefact can also be referred to as “mash.” The products of thisliquefaction can be concentrated and purified for food and otherapplications such as cleaning agents, textile agents, and animal feed.Herein, the term “biofuels” refers to any fuel derived from harvestedplant parts. Biofuels comprise but are not limited to biodiesel,vegetable oils, bioalcohols (i.e. ethanol, methanol, propanol, butanol,etc.) and biogases (i.e. methane).

In one embodiment, the liquefact is further processed to produceethanol. In one embodiment, the use of at least two different classes ofα-amylase enzymes in the liquefaction process results in a substratethat leads to higher ethanol yields compared to the ethanol yield fromstarch-containing plant material that is exposed to only one class ofα-amylase enzymes. When comparing the yield of ethanol from liquefactresulting from hydrolysis of starch-containing plant material exposed tovarious combinations of α-amylase enzymes, the comparison is performedwithin the same DE range for each “test” and “control” condition. Forexample, when comparing the yield of ethanol using a single class ofα-amylase enzyme (“control”) to the yield of ethanol using two differentclasses of amylase enzymes (“test”), the concentration of each class ofα-amylase enzymes in the test group is adjusted to obtain a similar DEunder similar hydrolysis conditions as the control α-amylase (whereconditions are otherwise adjusted for optimum performance of each classof enzyme). See, for example, Experimental Example 3. In one embodiment,the comparison is performed at a DE between 10 and 15. Such an increasemay be considered to be synergistic. For the purposes of the presentinvention, a “synergistic” increase in ethanol production is anyincrease in ethanol production that is higher than the ethanolproduction of either individual class of α-amylase alone, when measuredwithin equivalent DE ranges. While not being bound to any particulartheory or mechanism, an increase in ethanol production may be the resultof the number or ratio of different types (i.e., variety) of starchhydrolysis products resulting from hydrolysis of starch-containing plantmaterial using at least two different classes of α-amylase enzymes.

In various embodiments, the increase in ethanol yield is at least about0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, atleast about 0.5%, at least about 0.6%, at least about 0.7%, at leastabout 0.8%, at least about 0.9%, at least about 1%, at least about 1.1%,at least about 1.2%, at least about 1.3%, at least about 1.4%, at leastabout 1.5%, at least about 1.6%, at least about 1.7%, at least about1.8%, at least about 1.9%, at least about 2%, at least about 3%, atleast about 4%, at least about 5%, at least about 6%, at least about 7%,at least about 9% or greater. Even small increases in ethanol yield willtranslate to large volumes of ethanol produced over time in acommercial-scale fermentation process. Such improvements in ethanolproduction could result in a significant increase in profit to theethanol producer.

In another embodiment, the methods disclosed herein result in a loweramount of residual sugar remaining after fermentation when compared tothe amount of residual sugar remaining after fermentation ofstarch-containing plant material that is exposed to only one class ofα-amylase enzymes. The amount of residual sugar is reduced by at leastabout 1%, at least about 2%, at least about 3%, at least about 4%, atleast about 5%, at least about 6%, at least about 7%, at least about 9%,at least about 10%, about 15%, about 20%, about 25%, at least about 30%or more. Residual sugar can be measured in terms of total carbohydratelevels or in terms of total glucose levels remaining after, for example,approximately 48 hours of fermentation.

Liquefaction

The methods used for liquefaction of an aqueous slurry ofstarch-containing plant material vary depending, in part, on the natureof the α-amylase enzymes used in the process as well as the downstreamuse of the intermediate and end products. The steps can involve a singleliquefaction step, or may involve a primary liquefaction, followed byjet cooking and then a secondary liquefaction. The term “secondaryliquefaction” refers to a liquefaction process that takes place after aninitial period of liquefaction or after a jet cooking step of amulti-stage liquefaction process.

The conditions under which each step is performed also depends on thenature of the enzymes employed. For example, various α-amylase enzymeshave different degrees of thermostability and different requirements forpH. The steps should be performed under conditions sufficient for eachclass of α-amylase to hydrolyze the starch-containing plant material.

As discussed supra, each class of α-amylase enzyme can be provided inthe slurry as transgenic plant material expressing one or both classesof α-amylase enzymes, may be provided as a purified orpartially-purified enzyme preparation and exogenously added to theslurry, or may be provided in any combination thereof. The slurry maycomprise an admixture of a first starch-containing plant materialexpressing at least a first class of α-amylase, and a secondstarch-containing transgenic plant material that expresses a secondclass of α-amylase. Alternatively, the different classes of α-amylaseenzymes can be expressed from a single variety of plant that expresseseach α-amylase through transformation or breeding. The admixture mayfurther comprise starch-containing plant material that does not expressan α-amylase enzyme. Likewise, the admixture may comprise transgenicstarch-containing plant material expressing only one class of α-amylaseenzyme (where the second class of α-amylase is added exogenously to theslurry) or may consist only of starch-containing plant material thatdoes not express an α-amylase enzyme (where each class of α-amylase isadded exogenously to the slurry). The slurry can further comprise anaqueous solution (e.g., water, de-ionized water, backset (i.e.,stillage), etc.).

In some embodiments, dry plant materials from differentstarch-containing plants are mixed together before wetting with theaqueous solution. In other embodiments, the different starch-containingplant materials are added sequentially or simultaneously to a vesselwhile an aqueous solution is being added.

Where one class of α-amylase is provided in transgenic plant material,and the other class of α-amylase is provided exogenously as a purifiedor partially-purified enzyme preparation, the initial liquefaction stepsmay be performed under conditions compatible with thetransgenically-expressed α-amylase. The exogenous α-amylase can be addedto the slurry during the initial liquefaction steps if the enzyme hassimilar thermostability and pH optimum characteristics as thetransgenically-expressed α-amylase. Alternatively, the exogenousα-amylase can be added in a secondary liquefaction step. This secondaryliquefaction step should be performed under conditions sufficient forthe exogenous α-amylase to hydrolyze the starch-containing material,which may or may not require adjustment of the pH and/or ionconcentrations of the slurry.

Where both classes of α-amylase are added to the slurry exogenously, theliquefaction steps should be compatible with both classes of enzymes,and may require separate liquefaction steps with adjustment of pH and/orion concentrations between the steps. One of skill in the art willrecognize that the pH, ion concentration, temperature, and length oftime for each step can be optimized according to the type of α-amylaseenzymes employed in the liquefaction as well as the products desiredfrom the liquefaction. Exemplary, non-limiting liquefaction methods areprovided below.

It is also contemplated that the different classes of α-amylase can beadded to the starch-conversion process simultaneously or sequentially,and each can be added at any step in the process. The invention is alsonot limited to any particular order in which the different classes ofα-amylase are added to the slurry or the liquefact. Where one class ofamylase enzymes is provided in a transgenic plant, and one or moreadditional classes of amylase enzymes is provided as a purified orpartially-purified enzyme preparation, the purified orpartially-purified enzyme preparation can be added to the transgenicplant material in the initial step(s) of liquefaction, and/or may beadded in a subsequent step (including during saccharification orfermentation). Where at least two classes of α-amylase enzymes areprovided as transgenic plant material, plant material expressing eachenzyme can be provided in the initial step(s) of liquefaction, or theplant material expressing one or more classes of α-amylase can beprovided in the initial step(s) and plant material expressing one ormore different classes of α-amylases can be provided in one or moresubsequent steps. The latter scenario is also contemplated in the eventthat two or more classes of α-amylase are provided as purified orpartially-purified enzyme preparations.

A common enzymatic liquefaction process involves adjusting the pH of astarch slurry to the pH optimum of the α-amylase employed in themethods, with the addition of calcium hydroxide, sodium hydroxide orsodium carbonate. The addition of calcium hydroxide has the advantage ofalso providing calcium ions which are known to stabilize α-amylasesagainst inactivation. Upon addition of α-amylase (either throughprovision of transgenic plant material expressing the α-amylase orexogenous addition of α-amylase), the suspension is pumped through asteam jet to instantaneously raise the temperature to between 80° C. to115° C. The starch is immediately gelatinized and, due to the presenceof α-amylase, depolymerized through random hydrolysis of a (1-4)glycosidic bonds by α-amylase to a fluid mass which is easily pumped.

In a second variation to the liquefaction process, α-amylase is added tothe starch suspension, the suspension is held at a temperature of80-100° C. to partially hydrolyze the starch granules, and the partiallyhydrolyzed starch suspension is pumped through a jet at temperatures inexcess of about 105° C. to thoroughly gelatinize any remaining granularstructure. After cooling the gelatinized starch, a second addition ofα-amylase can be made to further hydrolyze the starch.

A third variation of this process is called the dry milling process. Indry milling, whole grain is ground and combined with water. The germ isoptionally removed by flotation separation or equivalent techniques. Theresulting mixture, which contains starch, fiber, protein and othercomponents of the grain, is liquefied using α-amylase. A practice in theart is to undertake enzymatic liquefaction at a lower temperature whenusing the dry milling process.

Typically, after gelatinization, the starch solution is held at anelevated temperature in the presence of α-amylase until a DE of 10-20 isachieved, usually a period of 1-3 hours. Dextrose equivalent (DE) is theindustry standard for measuring the concentration of total reducingsugars, calculated as D-glucose on a dry weight basis. Unhydrolyzedgranular starch has a DE of virtually zero, whereas the DE of D-glucoseis defined as 100.

The heating step of the liquefaction process can also involve the use oftemperatures below those used during conventional liquefaction processes(e.g., below about 95° C. to 120° C.). In some embodiments, the firsttemperature ranges from about 60° C. to about 85° C. In someembodiments, the first temperature ranges from about 75° C. to about 80°C. In other embodiments, the liquefaction does not include a jet-cookingstep. In some embodiments, the liquefaction does not include a secondaryliquefaction step. Thus, the presently disclosed liquefaction method, insome embodiments, involves a single heating step, or no heating step atall.

In various embodiments, the period of time for each liquefaction step isless than about 180 minutes. In some embodiments, the period of timeranges from about 20 minutes to about 35 minutes. In some embodiments,the first period of time ranges from 22 minutes to 30 minutes (e.g., canbe about 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes).

At each step, the slurry will have a pH that is optimal for theparticular α-amylase being employed in that step. Certain α-amylaseenzymes are known to function optimally at the natural pH of the slurry(i.e., the pH of the mixture of starch-containing plant material, water,and/or backset). Thus, this slurry can be used without the addition ofany pH adjusting chemicals. In some embodiments, this slurry has a pH ofbetween about 4.5 and about 5.2. In some embodiments, the pH is about4.8.

In some embodiments, jet cooking could be desired. Thus, in someembodiments, the process further comprises a jet cooking step followingthe heating step. In some embodiments, the jet cooking comprises heatingthe slurry to a temperature ranging from about 90° C. to about 120° C.for a period of time ranging from about 3 minutes to about 15 minutes.

In order to facilitate wetting or mixing the aqueous slurry, theliquefaction process can include an initial step of holding the slurryin a tank (i.e., a pre-slurry tank) for a period of time prior to theheating step. Any suitable mixing method can be used, including anysuitable manual or mechanical mixing method that can be used inconjunction with the pre-slurry and slurry (i.e., liquefaction) tanks.If the slurry is prepared in a separate tank or vessel than that inwhich the heating will take place, the slurry can be moved to theheating tank by any suitable approach (e.g., pouring, pumping, or thelike).

In some embodiments, the starch liquefact prepared from the presentlydisclosed process has a dextrose equivalent (DE) of at least about 13.In some embodiments, the DE of the liquefact is between about 10 andabout 20, or between about 13 to about 17. Thus, the DE of the liquefactcan be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

Ethanol Production

Also provided herein are methods for producing ethanol comprisingliquefying an aqueous slurry of starch-containing plant material in thepresence of at least a first and a second α-amylase enzyme, wherein thefirst α-amylase enzyme exhibits a starch hydrolysis pattern that isdifferent from the starch hydrolysis pattern of at least the secondα-amylase enzyme to obtain a liquefact; and fermenting the liquefact byyeast to obtain ethanol.

Prior to fermentation, the starch liquefact can be sacchrafied. Thesaccharification can include adding one or more starch-digesting enzymesto the starch liquefact during liquefaction (simultaneous liquefactionand saccharification, SLS) or after liquefaction. In some embodiments,the additional starch-digesting enzymes include glucoamylase.Glucoamylases (α-1,4-glucan glucohydrolases, E.C.3.2.1.3.) are starchhydrolyzing exo-acting carbohydrases. Glucoamylases catalyze the removalof successive glucose units from the non-reducing ends of starch orrelated oligo and polysaccharide molecules and can hydrolyze both linearand branched glucosidic linkages of starch (amylose and amylopectin).

The amount of glucoamylase employed in the present process can varyaccording to the mixture of dextrins present in the starch liquefact.For example, if the starch liquefact is high in concentration offermentable, small sugars, less glucoamylase might be needed. Theglucoamylase can be provided as transgenic plant material in the initialslurry, or can be added exogenously, or both.

The saccharification process can further include a heating step, whereinthe starch liquefact comprising additional starch-digesting enzymes(i.e., the saccharification mixture) is heated to a temperature (e.g., atemperature that allows for optimal activity for the enzymes employed)for a period of time. For example, the starch liquefact can be heated inthe presence of an additional starch-digesting enzyme (e.g.,glucoamylase) for a period of time from about 5 minutes to about 90minutes at a temperature from about 60° C. to about 75° C. Thetemperature can be chosen to be compatible with thermostableglucoamylases, such as those derived from Thermomyces lanuginosus (i.e.,T1GA).

In some embodiments, the heating step effects complete saccharificationof the slurry. Thus, in some embodiments, approximately 100% of theglucose expected from hydrolysis of the starch in the slurry is producedduring the heating step.

The glucose produced from a complete SLS process can be recovered by anysuitable approach. In addition to glucose, the heated slurry cancomprise additional materials, such as oil, protein and fiberby-products of the SLS process. These materials can also have economicvalue and can be recovered, as well.

The recovered glucose can be prepared in any formulation suitable forfermentation (e.g. to alcohol). The amount of recovered glucose fed intoa fermentor can be controlled so as to enhance the survival of the yeastin the fermentor. Alternatively, the glucose can be used in otherproducts, e.g. as a sweetener, in sweetened foods, for intravenoussolutions for use in hospitals or other medical settings. The glucosecan also be used to prepare other chemicals. For example, the glucosecan be treated with glucose isomerase to prepare fructose.

In some embodiments, the heating step effects partial saccharificationof the slurry. For example, heating can lead to a mixture containing atleast some glucose and some larger dextrins. In some embodiments, theheating step can yield about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or10% of the theoretical amount of glucose expected from completesaccharification based upon the amount of starch initially present inthe slurry. In some embodiments, the heating provides about 10%saccharification of the slurry. Thus, in some embodiments, the partialsaccharification can provide about 10% of the expected amount of glucoseexpected based upon the amount of starch in the slurry.

Subsequent fermentation of a partially saccharified mixture can beadvantageous, in that it allows for control of the initiation, rate,and/or extent of fermentation activity during a SSF process. Inparticular, the quantity of different starch-containing plant materialsand or enzymes can be adjusted to provide a suitable amount of glucoseto enhance the survival of yeast during a subsequent fermentation of themixture resulting from the SLS process. The amount of glucose being fedinto a fermentation process can also affect the quality of co-productsof the fermentation process, including DDG and DDGS. Alternatively, theglucose resulting from a SLS process can be removed from the mixture andused for any desired purpose.

In some embodiments, the process can include a separate secondsaccharification step. For example, the process can comprise heating themixture containing glucose to a second temperature for a second periodof time, thereby effecting complete saccharification of the mixture.This second heating step can include the addition of additional enzyme,e.g., additional glucoamylase.

In some embodiments, the mixture from a partial SLS process can be usedin a fermentation wherein additional saccharification can take placeduring fermentation. Thus, in some embodiments both additional enzymesand yeast can be added to the mixture, thereby producing additionalglucose and producing ethanol. In some embodiments, the glucose of thepresently disclosed process can be used to produce an end productselected from the group consisting of an alcohol, lactic acid, an aminoacid, fructose, citric acid, propanediol, DDG, DDGS, or a combinationthereof.

Following liquefaction, saccharification, or SLS, the resultinghydrolyzed sugars and starch are fermented. In some embodiments, thefermenting involves a simultaneous saccharification and fermentation(SSF) step. In yet another embodiment, the starch-containing plantmaterial may be used in raw starch fermentation. In the raw starchfermentation, the starch is not liquefied before enzymatic hydrolysis,and the hydrolysis is carried out at a temperature below gelatinizationsimultaneously with the fermentation process. In one embodiment theinvention may be useful in a bacterial or yeast fermentation whereinproducts from starch hydrolysis are desired. In one embodiment theinvention may be useful in a cell free fermentation as described inAllain et al. Journal of Chemical Technology and Biotechnology 82:117-120 (2007).

In some embodiments, the fermenting comprises adding a solution of yeastto the cooled starch liquefact or raw starch and agitating the cooledstarch liquefact at temperature from about 28° C. to about 35° C. for aperiod of time sufficient for conversion of a sufficient quantity of thesugars to ethanol, e.g., from about 12 to about 72 hours. In someembodiments the yeast is Ethanol Red yeast.

The saccharification and/or fermentation mixture can include additionalingredients to increase the effectiveness of the process. For example,the mixture can include added nutrients (e.g., yeast micronutrients),antibiotics, salts, added enzymes, and the like. Nutrients can bederived from stillage or backset added to the liquid. Suitable salts caninclude zinc or magnesium salts, such as zinc sulfate, magnesiumsulfate, and the like. Suitable added enzymes include those added toconventional processes, such as protease, phytase, cellulase,hemicellulase, exo- and endo-glucanase, xylanase, and the like. In someembodiments, the process comprises adding one or more reagents from thegroup consisting of an additional starch-digesting enzyme, a yeastextract, an antibiotic, and yeast to the starch liquefact.

The product of the fermentation process can be referred to herein as“beer”. For example, fermenting corn produces “corn beer”. Ethanol canbe recovered from the fermentation mixture (i.e., from the beer) by anyof a variety of known processes. For example, ethanol can be recoveredby distillation. Thus, in some embodiments, the presently disclosedprocess further comprises an ethanol recovery step. This step cancomprise distillation.

The remaining stillage includes both liquid and solid material. Theliquid and solid can be separated by, for example, centrifugation. Therecovered liquid, thin stillage, can be employed as at least part of theliquid for forming the saccharification and fermentation mixture forsubsequent batches or runs.

The recovered solids, often referred to as distiller's dried grain(DDG), include unfermented grain solids and spent yeast solids. The thinstillage can be concentrated to a syrup, which can be added to the DDGand the mixture then dried to form distiller's dried grain plus solubles(DDGS). DDG and/or DDGS can be sold as animal feed.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1: Method of Determining the Hydrolysis Pattern andAmount Using an Enzyme Extract

Using alpha amylases from various sources, a series of lab scaleliquefaction reactions were conducted using conditions similar to thoseused in modern ‘dry grind’ ethanol plants. In the industrial processwherein the alpha amylase as add as a liquid enzyme, ground corn mealare combined with water and a thermotolerant alpha amylase enzymes and,with continuous agitation, the mixtures are heated to approximately 85°C. and held for a period during which the starch is converted to sugarsand dextrins of a variety of molecular weights and conformations.Alternatively, when corn amylase is a component in an industrialprocess, ground corn meal wherein at least a component of the corn mealcontains alpha amylase are combined with water and the mixtures areheated as described previously. An instrument from the Mathis company,called the Labomat, was used in this laboratory simulation of industrialliquefaction to control the reaction and maintain constant mixing and atemperature of 85° C.

During the liquefaction reaction the starch within the corn wasgelatinized and then hydrolyzed by the various alpha amylase enzymes.The varying degrees and types of hydrolysis products were obtained bythe combined effects of dose and type of enzyme used. The analysis ofthe oligomer profile is analyzed after enzymatic digestion at hightemperature and subsequent cooling of the reaction to between 25-30° C.The group of carbohydrate products being evaluated in these experimentsare produced by the catalyzed breakdown of starch in a flour of wholecorn, followed by the potential formation of retrograde ortransglycosylated carbohydrate products created during cooling orthrough the action of other enzymes present in the corn or enzymeextract. Following the liquefaction reactions a variety of techniqueswere employed to evaluate the types and relative amounts of sugarsliberated from each condition or combination of enzymes.

High Temperature Liquefaction of Ground Corn

After determining the percent moisture of the ground corn, a mixture ofcorn, water and enzyme was prepared to achieve a mixture with a percentof dry solids of 33.5%. The enzymes used may be derived from varioussources. For example as previously described an amylase from a Bacillusorganism may be used or an alpha-amylase enzyme derived from, containingprotein domains from, or sharing sequence homology with a proteinisolated from a microorganism of the phylogenic order Themococcales.Alpha-amylases for example, such as those described in US PatentPublication US2003/0125534 and US2004/0018607 such as D45, BD12870 or797GL3 may be further characterized in regards to their respectivestarch hydrolysis pattern and be further employed in starch hydrolysisas described herein. These enzymes may be in crude, partially-purifiedor purified form and exogenously added to a liquidfaction.Alpha-amylases may also be expressed transgenically in a plantessentially as described in Example 3 for 797GL3. Alternatively, D45 orBD12870 DNA sequence can be operably linked to the 27 kD gamma-zeinpromoter in order to direct expression of the enzyme to endosperm of theseed. In addition, a gamma-zein targeting sequence and an endoplasmicreticulum (ER) retention signal (SEKDEL) can be used to retain theenzyme in the ER organelle. The above described expression cassette canthen be incorporated into an agrobacterium transformation vector andtransgenic corn plants produced by agrobacterium transformation asdescribed in US 2003-0135885.

A Bacillus enzyme was used, including 2.5 μl/gram corn, 5 μl/gram, 7.5μl/gram, and 10 μl/gram. A total of 200 grams of corn was used to ensurea percent of dry solids of 33.5%. The mixture was then agitatedthoroughly to distribute the enzyme and mix the water with the cornflour. The individual reactions with a unique combination of enzyme doseand type were added to 1 L stainless steel beakers used to process thereactions. A Mathis Labomat equipped with a 16 position carousel wasused to process the liquefaction reactions. The instrument wasprogrammed to heat the reactions at 6° C./minute and then hold at 85° C.for 1 hour with a continuous rotation of the beakers at 65 rpm, changingdirection of rotation every 60 seconds. After liquefaction at hightemperature, the reaction is allowed to cool to temperatures between25-30° C. before analysis for oligomer profiles/concentrations andsubsequent fermentation analysis.

The cooked corn slurry was then analyzed using HPLC techniques and theFehling's assay to characterize the hydrolysis of starches. The resultsfor varying concentrations of the Bacillus licheniformis Type XII-Aenzyme (Sigma) are shown in Tables 2-5.

Percent Moisture Determination

Using the Mettler HB-43 Moisture Balance the moisture content of avariety of samples can be determined. The majority of samples needingmoisture measurement are ‘dry’ powders. These samples require littlespecial care to determine moisture. However there are special methodsthat must be followed to achieve the optimal measurements when runningsamples with a moisture content exceeding 15%. The moisture content ofcorn flour liquefactions exceeds 60% and has moderate concentrations ofsugars and oils that may degrade under high temperature, thereforeprocessing will require the use of supplementary tools. Using glassfiber or other fiber discs from Mettler or Ohaus, moisture will bewicked away from the sample to increase evaporation rate. Lowtemperatures, <105° C., are to be used for drying samples with highmoisture contents or temperature-sensitive samples. Otherwise corn flourand other ‘dry’ solids can be measured using temperatures up to 130° C.All samples used for moisture determination are terminal samples andshould be discarded after use. For more details about the instrument andits operation refer the manufacturer's literature.

The “switch-off criterion” is set to the value of “5” before anymeasurements are obtained. This switch-off criterion is based on aweight loss per unit of time. As soon as the mean weight loss is lessthan a preset value during a specified time, the instrument considersdrying as complete and automatically discontinues the measurementprocess. During the drying, the time display shows how long themeasurement process has been in progress; the switch-off criterion isinactive during the first 30 sec.

The moisture balance should be set to heat samples at the correct dryingtemperature. The drying procedure should take between 30 and 40 minutesfor slurries at ˜105° C. Dry powders usually take 6-8 minutes at 130° C.A 0.75+/−0.01 gram sample of the dry powder is distributed evenly acrossa clean aluminum pan set in the balance to get an accurate moisturemeasurement. For slurries, a 10 mL pipette is used to evenly spacedroplets directly on the aluminum pan totaling 6-8 grams. These dropsare then covered lightly by either the filter disc or glass wool and themeasurements obtained.

Preparation of Samples for HPLC Analysis

A 50 ml sample of each slurry is placed in a new 50 ml conical bottomedtube. The tubes are centrifuged for 10 minutes at 3,500 rpm. Followingthe spin, 1.5 ml of the supernatant is removed with a large pipette andadded to a new 1.8 ml microcentrifuge tube. The samples are then spunfor 10 minutes at 14,000 rpm. The supernatant is then transferred to aCostar Spin-X column and spun in a microcentrifuge for 5 minutes at5,000 rpm. The filtrate is diluted 1:5 by volume with pure water andused for analysis in HPLC.

Size Exclusion Chromatography

The Waters Alliance HPLC system was used to analyze samples with atechnique known as size exclusion chromatography. The general method isflexible and can be adapted to a variety of instruments equipped withother components and conditions. The specific method used for thisanalysis enabled the separation of soluble sugars produced duringliquefaction using the Waters Ultrahydrogel 250 column. The columnseparated sugars based on size with the largest columns being elutedfrom the column first. Refractive Index changes are used to produce asignal that is correlated to the concentration of sugars of varioussizes being eluted from the column. The instrument was operated with amobile phase of water at a flow rate of 0.8 ml/minute with the column ata temperature of 45 C and the Refractive Index detector at a temperatureof 45 C. An injection of 10 μl of each diluted sample was injected bythe instrument and the refractive index monitored over a period of 20minutes. To estimate the amount of general size of sugars being eluted,a series of sugar standards at known concentrations were processed bythe instrument. These standards included solutions of DP1, DP2, DP3,DP4, DP5, DP6, DP7 and dextrins ˜DP32, 147 and 300.

Sugar-5 Chromatography

The Waters Breeze HPLC system was used to analyze samples with atechnique used currently by industrial ethanol producers to evaluate theamounts of low molecular weight sugars, organic acids, glycerol andethanol produced during the conversion of grains to ethanol. The generalmethod is flexible and can be adapted to a variety of instrumentsequipped with other components and conditions. The specific method usedfor this analysis used a Biorad Aminex HPX-87H Ion Exclusion column forthe separation of the aforementioned constituents. Refractive Indexchanges are used to produce a signal that is correlated to the type andconcentration of compounds being eluted from the column. The mobilephase of 5.0 mM was pumped at a rate of 0.6 ml/minute with a columntemperature of 60 C and the Refractive Index detector at a temperatureof 45° C. An injection volume of 25 ul of each sample (diluted or not)was injected by the instrument and the refractive index monitored over aperiod of 30 minutes. To estimate the amount and type of compounds beingeluted, a series of mixed standards were prepared that contained sugars,dextrins, acetic acid, lactic acid, glycerol and ethanol at knownconcentrations and were processed by the instrument. The instrument wascalibrated using these standards and quantitation achieved by comparisonto the linear regression line established for each compound.

Dextrose Equivalent

Standardization of Copper-Tartrate Solution and Preparation of Blank

Exactly 1.0000+0.0001 g of oven-dried dextrose is weighed andtransferred quantitatively to a 100-mL volumetric flask, and diluted tovolume with RO water. One sample is prepared by adding 5 mL of the 1%glucose solution to a 500-mL beaker containing 20 mL water. Then a blankis prepared with 25 mL of water in a 500 mL flask. Fehlings Solution A(10.0+0.1 mL) is accurately measured and added into each beaker. ThenFehlings Solution B (10.0+0.1 mL) is accurately measured and added intoeach beaker. Each beaker is swirled to mix thoroughly. Several glassbeads are placed into each beaker. Each beaker is heated on a hot plateuntil the solution boils for 3-4 minutes. Each beaker is removed fromthe hot plate and cooled in a cold tap water bath. After cooling,10.0+0.1 mL KI solution and 10.0+0.1 mL of H2SO4 solution are added, inorder, and the beakers are swirled. Each sample is titrated with 0.1Nsodium thiosulfate solution until a light straw color is reached. Twomilliliters of starch indicator is added and titrated until the sampleturns a milky white. The titration volume is recorded.

Sample Assay—Manual Titration

When determining the DE for a liquefied corn flour sample it isnecessary to use the correct amount of sample so as not to overload theassay. This amount is expressed in terms of mass of dry carbohydrate.When using Maltrin or other pure carbohydrate samples, the moisturecontent is determined using a moisture balance and the mass added to theassay is corrected for that moisture value. The corn flour liquefact isdiluted before a sample is taken for use in this method. To determinethe amount of a corn or carbohydrate sample needed to obtain an accurateresult from the Fehling's assay, the protocol in Appendix I is used.

A 500-mL flask is placed on the analytical balance and the balancecalibrated. The sample is weighed into a beaker and the weight recordedto 4 decimal places. (Table 1 lists mass of carbohydrate for given DEranges of dried product). Three beakers are prepared for Maltrin MD105,150 and 250 maltodextrins. These serve as internal controls to monitorassay performance.

Enough reverse osmosis (RO) water is added to bring the total volume toapproximately 25 mL. The beaker is swirled to ensure that the sample iscompletely dissolved/mixed. Fehlings Solution A (10.0 mL) is accuratelymeasured and added into each beaker. Then Fehlings Solution B (10.0 mL)is accurately measured and added into each sample flask. Each beaker isswirled to mix thoroughly. Several glass beads are placed into eachbeaker. Each sample beaker is heated on a hot plate until the solutionboils for 3-4 minutes. (Approximately 7 minutes total time) Each beakeris removed from the hot plate and cooled in a cold water bath. Aftercooling, 10.0+0.1 mL of the KI solution and 10.0+0.1 mL of the H₂SO₄solution are added, in order, and the beakers are swirled. Each sampleis titrated with 0.1N sodium thiosulfate solution until a light strawcolor is reached. 2 mL of starch indicator is titrated into the sampleuntil the sample turns a milky white. The titration volume is recorded.

Calculation of Dextrose Equivalent:

Two calculations are included for the determination of DE from theFehling's method:

${{Dextrose}\mspace{14mu} {Equivalent}\mspace{11mu} \left( {{Carbohydrate}\mspace{14mu} {Only}} \right)} = \frac{500 \times \left( {{\Delta \mspace{14mu} {titrate}\mspace{14mu} {of}\mspace{14mu} {blank}} - {\Delta \mspace{20mu} {titrate}\mspace{14mu} {of}\mspace{14mu} {sample}}} \right)}{\begin{matrix}{\left( {{Dry}\mspace{14mu} {Carbohydrate}\mspace{14mu} {mass}\mspace{14mu} {gm}} \right) \times 100 \times} \\\left( {{\Delta \mspace{14mu} {titrate}\mspace{14mu} {of}\mspace{14mu} {blank}} - {{titrate}\mspace{14mu} {of}\mspace{14mu} {standard}}} \right)\end{matrix}}$${{Dextrose}\mspace{14mu} {Equivalent}\mspace{11mu} \left( {{No}\mspace{20mu} {Carbohydrate}\mspace{14mu} {Correction}} \right)} = {{\frac{500 \times \left( {{\Delta \mspace{14mu} {titrate}\mspace{14mu} {of}\mspace{14mu} {blank}} - {\Delta \mspace{20mu} {titrate}\mspace{14mu} {of}\mspace{14mu} {sample}}} \right)}{\begin{matrix}{\left( {\left( {{Total}\mspace{14mu} {dry}\mspace{14mu} {mass}\mspace{14mu} {in}\mspace{14mu} {grams}} \right) \times 100} \right) \times} \\\left( {{\Delta \mspace{14mu} {titrate}\mspace{14mu} {of}\mspace{14mu} {blank}} - {\Delta \mspace{20mu} {titrate}\mspace{14mu} {of}\mspace{14mu} {standard}}} \right)\end{matrix}}*{dry}\mspace{14mu} {{carbo}.\mspace{14mu} {mass}}} = {\left( {{total}\mspace{14mu} {dry}\mspace{14mu} {mass}} \right) \times \left( {\% \mspace{14mu} {starch}\mspace{14mu} {composition}\mspace{14mu} {of}\mspace{14mu} {sample}} \right)}}$

APPENDIX I

This assay will determine the amount of reducing ends in a sample thatcontains carbohydrates. To calculate DE, the percentage of carbohydratescontained within a sample is required to determine the correct value.There is a maximum amount of reducing ends that can be present in asample while providing accurate and useful data using the Fehling'smethod. A sample that is mostly small sugars will have a high DE valueand will require significantly less mass to fall within the workingrange of this assay when compared with a sample that has mostly highmolecular weight sugars, maltodextrins or small starch molecules. If thesample is suspected to contain mostly small sugars and is not dilutedproperly then the assay will be saturated before titration begins.

Table 1 provides a quick reference to determine the correct dry samplemass for use in the Fehling's method by using an estimate of the DEvalue. For samples with unknown DE values, a very small mass ofapproximately 0.1 g is used for initial titrations. When using a purecarbohydrate sample, the mass of dry sample to use in this method isobtained from column 2 of Table 1. If using corn or other types of grainflour column 3 provides the correct mass of a sample to use.

The procedures in this protocol have been modified to measure DE fromcorn flour liquefactions (cooked wet slurries at 20 to 35% dry solids).Some liquefacts are hydrolyzed to DE's in excess of 30 and therefore avery small sample can be used effectively. Through employment of adilution scheme, a solution with corn flour can be obtained insuspension where the equivalent of 0.1-1 grams of dry flour is presentin a given volume, usually 3-5 mL, and then added to the assay to keepthe sample within the dynamic range. The corn flour liquefact should bediluted 1:20 by mass to achieve a solution that will be dilute enough toallow for the determination of DE values>25. If the 1:200 dilution isused for this assay up to 25 mL of the dilution can be used as a sample.

A 10 gram dry flour sample is mixed with water or other additives andliquefied. The percent solids or percent moisture is determined and usedto determine what the 10 gram sample of flour now weighs in the presenceof water. For example, if 10 grams of flour was liquefied at 30% solidsthe mass of a 10 gram dry flour equivalent of liquefact is (10 g dryflour*100)/30=33.3 g. Therefore 33.3 grams of the wet liquefact at 30%solids represents 10 g of dry flour. This 33.3 grams will then bediluted to 200 grams with water. This represents an effective 1:20dilution of corn flour [(10 g dry flour)/(166.7 g of H2O+33.3 g cornslurry)]. From this 1:20 dilution an aliquot of the suspension is takenand used directly in the Fehling's method. The beaker in which the assayis run is zeroed on a balance. To the beaker is added enough dilutedliquefact to equal the equivalent of 0.1-1.0 g of dry flour massdepending of expected DE utilizing Table 1.

It is important to determine the true mass of corn flour or othercarbohydrate source going into the assay so a balance capable of readingto the 0.0001 g is required. From the dilution, up to 25 mL can be usedbut the mass must be determined very accurately to back calculate themass of corn flour. For standards or dry compounds, the mass can beweighed directly into the beaker, skipping the dilution series.

TABLE 1 Mass of Substance For Use in the Fehling's Method Dry mass ofpure Dry mass of Expected DE carbohydrate (g) corn flour (g) 4-7 0.901.29  8-12 0.45 0.64 13-17 0.30 0.43 17-20 0.22 0.31 20-23 0.20 0.2923-27 0.16 0.23  26-100 0.10 0.14

Useful Equations for Corn Slurry Liquefact:

Dry mass of flour present in wet corn flour liquefact slurry at (x) %dry solids=Dry flour mass=[(Wet wgt. of liquefact)×(% dry solids ofliquefact)]/100

Mass of wet liquefact that represents (x) g of dry flour at (x) % drysolids=Wet wt. of liquefact=[(Dry flour wgt.)×100]/percent dry solids ofliquefact

Total dry mass=diluted sample mass (gm)/dilution factor

Dry carbohydrate mass (gm)=Total dry mass (gm)*% starch composition

Post-Liquefaction Oligosaccharide Distribution:

Size exclusion chromatography on liquefacts from Corn Amylase (797GL3)(described in Example 2) and commercially available α-amylases clearlydemonstrate the novel action pattern of both 797GL3 and D45 vsconventional amylases derived from bacterium of the Bacillus genus. BothCorn Amylase (797GL3) and D45 hydrolyze starch to smaller fragments thanBacillus amylases. The consequence of the different breakdown pattern ismanifested primarily during saccharification-fermentation.

The ethanol industry typically will use the Fehling's assay orequivalent test to determine the Dextrose Equivalent (DE) for the slurryproduced during the liquefaction process. Using liquid α-Amylase enzymesa producer will try to generate corn slurry that has a DE between 10 and15. When the a slurry with a DE of between 10 and 15 is analyzed usingSize Exclusion Chromatography the pattern of various sized sugars anddextrins generated by the enzyme can be visualized as it elutes from thecolumn.

The pattern produced using α-Amylases derived from bacterium of theBacillus genus are very similar to each other and very different fromthose produced using either 797GL3/Corn Amylase or D45 (FIG. 1 ofAtichokudomchai et al. (2006) Carbohydrate Polymers 64:582-588).Liquefied corn produced by liquefaction with Bacillus α-Amylasestypically have a substantial fraction of products that range from DP32to DP300 where liquefacts derived from Corn Amylase have almost nosugars in this size range. A bimodal molecular weight distribution hasbeen observed when analyzing hydrolysates of starch using Bacillus alphaamylase compared to as unimodal distribution observed when using CornAmylase.

Dionex Ion Exchange Chromatography

Ion exchange chromatography was performed according the MacGregor et al.(1994) Carbohydrate Research 2.57; 249-268, which is herein incorporatedby reference in its entirety.

Differences exist between oligomer profile patterns generated fromliquefacts produced with either Bacillus amylases or Corn Amylase. TheBacillus amylases produce a pattern with clearly defined and evenlyspaced peaks, while not to be limited by theory, the evenly spaced peakslikely representing linear chains of oligomers and also possess a singlepeak of rather high but unknown molecular weight. The pattern producedwith Corn Amylase alone shows a very different pattern with manyintermediate peaks and a lack of the high molecular weight peaks. SeeFIG. 4. This pattern is similar to the results shown for the proposedreaction pattern described in Atichokidomachai et al. (see FIG. 5therein) demonstrating differences in products from Bacillus amylasesand D45.

Example 2. Method of Making Corn Amylase

The terms corn amylase, CA, and corn amylase (797GL3) refer to cornplants expressing the α-amylase enzyme 797GL3. The enzyme is describedin US Patent Publication US2003/0125534 and US2004/0018607, which areherein incorporated by reference in their entirety. Methods of makingcorn plants expressing 797GL3 is described in US 2003-0135885. Asdescribed similarly in US 2003-0135885, corn amylase comprises anexpression cassette wherein a nucleotide sequence encoding the enzyme,797GL3, was operably linked to the 27 kD gamma-zein promoter in order todirect expression of the enzyme to endosperm of the seed. In addition, agamma-zein targeting sequence and an endoplasmic reticulum (ER)retention signal (SEKDEL) was used to retain the enzyme in the ERorganelle. The above described expression cassette was incorporated intoan agrobacterium transformation vector and transgenic corn plantsproduced by agrobacterium transformation as described in US2003-0135885.

Example 3. Use of Two Thermotolerant α-Amylases During Liquefaction toImprove the Production of Ethanol with Saccharomyces Cervisae and Corn

Milled corn was liquefied for 1 hour at 85 C in the Mathis labomat at pH5.8 at 33% dry solids. No thin stillage or other adjuncts were added tothe liquefact. Varying combinations of alpha amylase enzymes were usedduring the process and the DE was kept near constant for all conditions.A ‘benchmark’ dose of Bacillus enzyme was determined to produce a DE of10-15, 0.1 μl/dry gram, and then used a control. The corresponding doseof Corn Amylase to produce a similar DE, 5% admix was also used as acontrol. Three combinations of Corn Amylase and Bacillus were used forthe evaluation, 0.09 μl/dry gram Bacillus+0.5% admix of Corn Amylase(797GL3), 0.05 μl/dry gram Bacillus+2.5% admix Corn Amylase and 0.01μl/dry gram Bacillus+4.5% admix Corn Amylase. For each condition, 200grams of flour were liquefied in large labomat beakers. Moisture wasdetermined by using a Mettler Moisture Balance set at 105 C for ˜20minutes until weight loss <0.01 g/min.

Following liquefaction the slurry was allowed to cool to roomtemperature. From each reaction vessel three replicate fermentationreactions were assembled. The nutrient source was a concentratedsolution of yeast extract dissolved in water with gentle heating. Theantibiotic used was tetracycline dissolved at 10 mg/ml in 95% Ethanol.The glucoamylase used was derived from Aspergillus niger and iscommercially available from Sigma, produced by Novozymes Corp. After allof the ingredients were added to a 100 ml glass bottle, the cap wastightly closed, shaken vigorously for 30 seconds and then the cap wasopened minimally to vent evolved gases. The fermentations were thenplaced in a 32 C walk in incubator for 72 hours.

Small amounts of the slurry were analyzed using the BCA method todetermine DE (Dextrose Equivalent) (Table 2). A small aliquot was usedfor HPLC analysis using the Sugar 5 method and Size ExclusionChromatography to quantify and qualify the amount and distribution ofsoluble carbohydrates. An aliquot was also analyzed using the Dionexsystem equipped with an ion exchange column to qualify individualsugars, branched and linear, up to DP40.

TABLE 2 DE determination via BCA assay Sample # 1 2 3 4 5 Sample labelBac 0.10 ul/g Bac 0.09 ul/g + Bac 0.50 ul/g + Bac 0.01 ul/g + 5% CA 0.5%CA 2.5% CA 4.5% CA Relative 177.718 186.832 208.043 278.926 272.31concentration of glucose (μg/mL) Initial 400.62 401.05 400.22 400.65400.49 dilution (mL) final dilution 100 100 100 100 100 ratio % DS 33 3333 33 33 mass of 60.44 60.45 60.81 60.45 61.06 slurry Flour (g) 19.945219.9485 20.0673 19.9485 20.1498 glucose (g/L) 0.03198924 0.033629760.03744774 0.05020668 0.049016 glucose (g) 1.281552933 1.3487215251.49873345 2.011530634 1.963034 starch (g) 14.21305008 14.2154016814.30005916 14.21540168 14.35885 [corn 71% starch] DE 9.0167340959.487748254 10.48061014 14.15036085 13.67125

Additionally a small amount of liquefact was spiked with a high dose ofGlucoamylase ˜2 ml/75 g and 0.1 gm Sodium azide and incubated for 144hrs at 32 C to observe the ability of each condition to saccharify.

Conditions using both amylases show an increase in the amount of ethanolproduced at 48 hours with the two controls (CA5.0% and Bac 0.1 ul/g)being essentially equal (Table 3).

TABLE 3 Ethanol production Bac Bac Bac Timepoint Bac 0.09 ul/g + 0.05ul/g + 0.01 ul/g + CA (in hours) 0.1 ul/g CA 0.5% CA 2.5% CA 4.5% 5.0% 00 0 0 0 0 23.5 15.583 15.757 15.711 15.662 15.666 36 17.189 17.26517.032 17.151 17.220 48 18.253 18.305 18.345 18.297 18.232 72 18.40618.454 18.476 18.478 18.439

Another technique used to evaluate the amount of consumed carbohydratesis to look at residual soluble sugars, again the combination of the twoamylases produces amount lower that the Bacillus alpha amylase but notquite as low as Corn Amylase (Tables 4 and 5). Total residual starch canbe measured as described in Xiong, Y et al, Journal of Animal Science63: 3861 or AOAC method 996.11 herein incorporated by reference.

TABLE 4 Total residual sugars Bac Bac Bac Timepoint 0.09 ul/g + 0.05ul/g + 0.01 ul/g + (in hours) Bac 0.1 ul/g CA 0.5% CA 2.5% CA 4.5% CA5.0% 0 29.727 26.9 26.96 26.887 26.894 23.5 2.811 2.695667 2.8626672.846667 2.672667 36 1.139 1.160333 1.125667 1.158 1.017 48 0.7566670.769667 0.702333 0.680667 0.665333 72 0.912 0.897 0.664333 0.5636670.564667

TABLE 5 Total glucose Bac Bac Bac Timepoint Bac 0.09 ul/g + 0.05 ul/g +0.01 ul/g + CA (in hours) 0.1 ul/g CA0.5% CA2.5% CA4.5% 5.0% 0 0.6890.672 0.692 0.647 0.626 23.5 1.589 1.291 1.255 1.179 1.110 36 0.1350.116 0.160 0.163 0.125 48 0.053 0.055 0.033 0.024 0.017 72 0.279 0.2510.216 0.192 0.184

Example 4. Measurement of Viscosity Using Two Thermotolerant α-AmylasesDuring Liquefaction

To characterize the effects two amylase liquidfaction on viscosity, aseries of experiments were carried out using a unimodal and bimodalamylase. Unimodal amylases comprised 797GL3, D45, and BD12870. Amylasesexhibiting a bimodal hydrolysis patterns consisted of amylase derivedfrom a Bacillus organism. A 30% total solid liquefaction sample from alarge dry grind ethanol plant was collected and frozen. The sample wastreated with a Bacillus amylase prior to collection. Upon time foranalysis, the samples were defrosted at room temperature and 4×40 mlaliquots were prepared in 50 ml conical tubes. Viscosity was measuredusing a RVA-4 viscometer (Newport Scientific). To measure viscosity, thesamples are first heated up in a water bath to 85° C. Following, 25grams of each sample is weighed out and placed into a viscometer vesseland capped. These samples are then placed into the Newport ScientificRapid Visco Analyzer (RVA) and the paddle speed is set to 160 RPM.Instrument settings are such that the samples will be heated to 85° C.and held for 5 minutes. RVA will then be ramped down over 5 minutes to32° C. Samples will then be held at 32° C. for 5 minutes. Total run timeis therefore 15 minutes. Viscosity data is collected throughout the 15minute time period. Table 6 demonstrates the data points at minimumviscosity at 2-4 minutes at 85° C. and maximum viscosity is acquiredbetween 12-14 minutes at 32° C.

The first tube, control #1, was raised to 85° C. and 25 grams of mash isweighed out into a viscometer vessel and measured for viscosity asdescribed above. The second tube, control #2 was heated at 85° C. for 90minutes, then acid stopped with 400 μl of 40% H₂SO₄, and tested forviscosity. The purpose of control #2 is to determine if any additionalliquid Bacillus amylase activity would remain during additional cookingof the frozen mash sample. The third tube, spiked CA protein, wasprepared by adding 450 μl of a purified corn amylase protein extract (1mg protein/ml of 20% ethanol) to achieve an approximate 10× dose. Thethird tube was raised to 85° C. for 90 minutes, then acid stopped with400 μl of 40% H₂SO4, and tested for viscosity. The forth tube, spikedD45 liquid amylase, was prepared by adding 30 μl of formulated enzyme toachieve a approximate 10× dose and measured for viscosity. The forthtube, spiked BD12870 liquid amylase, was prepared by adding 500 μl offormulated enzyme. A drop in viscosity is observed for those sampleswhere a unimodal amylase has been added to the liquefact containing aBacillus amylase (See Table 6 for viscosity results).

TABLE 6 Viscosity measurements Sample MIN AT 85° C. MAX AT 32° C.control 1 197 442 control 2 183 366 450 uL CA 126 238  30 uL D45 116 229500 uL BD12870 149 298

Example 5. Additional Assays for Measuring Starch Liquefaction

To further characterize the unique hydrolysis patterns associated witheach individual group of alpha amylase enzymes, either those fromBacillus bacterium or Corn Amylase, and those created through the use ofcombinations of members of the two groups, a series of liquefactionexperiments are performed. Other amylases such as BD12870,Thermococcales derived amylases, unimodal or bimodal amylases may becombined essentially as described in Example 3 to show a synergisticdual mode of action benefit.

Using available alpha amylase enzymes and ground corn, amylose,amylopectins and purified corn starch as substrates, hydrolysis isperformed using conditions that are similar to those used in ethanolproduction facilities. Additionally, conditions that are not currentlybeing used for industrial processes can be utilized, especially onesthat may offer additional process efficiencies or yield advantages notattainable through the use of a single enzyme. These conditions mayinclude liquefaction at pH<5.8, very short liquefaction times <1 hour,lower temperature of liquefaction, the elimination of the jet cookingprocess, liquefaction of slurry that has a very high percent of drysolids (e.g., >33.5%). The same general protocol for generatingliquefact at a lab scale as described previously is used. To furthervalidate the synergistic effects of using two amylases of divergenthydrolytic action patterns, samples produced from a full scaleindustrial plant can be analyzed to compare the performance of the twoenzymes alone or in concert with results obtained from lab scaleexperiments.

For each of these liquefacts the degree of hydrolysis is measured usingthe Fehling's assay or equivalent technique to determine DextroseEquivalent. Additionally liquid chromatographic analysis is used tocharacterize the profile of oligomers generated through hydrolysis, bothquantitatively and qualitatively. Samples from large scale dry ethanolfermentations may also be collected and analyzed as described byExamples 1-6.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A method for producing a biofuel comprising:a) liquefying an aqueous slurry of starch-containing plant material inthe presence of at least a first and a second class of α-amylaseenzymes, wherein the first class of α-amylase enzymes exhibits a starchhydrolysis pattern that is different from the starch hydrolysis patternof at least the second class of α-amylase enzymes to obtain a liquefact;and, b) fermenting the liquefact to produce said biofuel.
 2. The methodof claim 1 further comprising a step of saccharification in the presenceof a glucoamylase, wherein the saccharification step can be performedsimultaneously with steps (a) or (b), or may be performed between steps(a) and (b).
 3. The method of claim 1, wherein the amount of biofuelobtained in step (b) is greater than the amount of biofuel obtainablefrom fermentation of hydrolyzed starch and sugars resulting fromliquefaction and saccharification of an aqueous slurry ofstarch-containing plant material in the presence of two or more classesof α-amylase enzymes that have similar starch hydrolysis patterns, or inthe presence of either the first or the second class of α-amylaseenzymes alone.
 4. The method of claim 1, wherein the amount of residualsugar remaining after step (b) is less than the amount of residual sugarremaining after fermentation of hydrolyzed starch and sugars resultingfrom liquefaction and saccharification of an aqueous slurry ofstarch-containing plant material in the presence of two or more classesof α-amylase enzymes that have similar starch hydrolysis patterns, or inthe presence of either the first or the second class of α-amylaseenzymes alone.
 5. The method of claim 1, wherein the biofuel is ethanol.6. The method of claim 1, wherein the fermentation is a yeastfermentation.
 7. A method for producing ethanol comprising raw starchfermentation of starch-containing plant material in the presence of atleast a first and a second class of α-amylase enzymes, wherein the firstclass of α-amylase enzymes exhibits a starch hydrolysis pattern that isdifferent from the starch hydrolysis pattern of at least the secondclass of α-amylase enzymes.